Steroid receptor regulated gene transcription is required for cell proliferation, differentiation and development and involves the formation of active transcription initiation complexes composed of a specific steroid receptor and a host of co-activators. The essential role of the androgen receptor (AR) in male sex differentiation is well established. Naturally occurring mutations result in the phenotypic spectrum of the androgen insensitivity syndrome characterized by incomplete male sexual development in utero. Two regions have been shown to be involved in steroid receptor transactivation: activation function 1 (AF1) in the NH2-terminal region and activation function 2 (AF2) in the ligand binding domain. In comparison to other steroid receptors, AF2 in AR is relatively weak reflecting its apparent reduced ability to recruit p160 coactivators through their LxxLL motifs. We have shown that a likely explanation for this unique property of the AR is the strong ligand (androgen)-mediated NH2-and carboxyl-terminal (N/C) interaction between an NH2-terminal FxxLF motif and the carboxyl-terminal AF2 hydrophobic surface. A second NH2-terminal N/C interaction site FxxLF interacts with the ligand binding domain in the absence of androgen binding. The hypothesis to be tested in the proposed research is that multiple intra- and intramolecular domain specific protein-protein interactions control ligand-AR mediated gene activation. The specific aims are to (1) identify the site within the ligand binding domain that interacts with the FxxLF NH2-terminal motif in the absence of androgen binding, (2) determine the roles of androgen and p160 co-activator binding in AR stabilization and identify the sequences that constitute the AR degradation signal, (3) identify the AR NH2-terminal interaction sites for p160 and other co-activators, determine which co-activators interact with AR transcription complexes in chromatin of androgen regulated genes, and test the requirement of p160 co-activators in AR transactivation using p160 co-activator gene knockout cell lines, (4) identify the AR site(s) that interact with PIAS1 (protein inhibitor of activated signal transducer and activator of transcription) and evaluate the physiological role of this interaction using AR mutations that cause androgen insensitivity, and (5) identify additional mutations and cellular factors that cause the androgen insensitivity syndrome in the absence of a mutation in the AR coding region. The studies will further define the domain interactions with AR and between AR and its co-activators and how these interactions are altered in the human androgen insensitivity syndrome.